Project Summary The goal of this project is to determine the timing and mechanism that govern type 1 alveolar epithelial cell (AT1) lineage specification in the developing lung. Formation of the architecturally complex gas exchange surface of the lung during early development requires precise temporal and spatial control of AT1 and type 2 alveolar cell (AT2) differentiation. Alterations in this process result in devastating developmental lung diseases. Despite our understanding of proximal airway developmental hierarchies, the distal alveolar ontogeny and mechanisms of specification are unclear. AT1 and AT2 lineages are morphologically distinct by embryonic day 17.5 and acquire highly specific functions. Thus, I hypothesize a critical phase exists before this stage, at which alveolar lineages become specified to differentiate. An AT1 cell-specific transcription co-repressor, Hopx, is first expressed, at E15.5. Hopx has been shown to repress alternative cell fate in cardiomyoblasts during development, and may play a similar role in repressing AT2 cell fate. We will test the hypothesis that AT1 lineage specification occurs at embryonic day 15.5, through Hopx-mediated repression of AT2 fate through the following aims: Aim 1 will elucidate the developmental ontogeny of AT1s by determining the developmental stage at which they become specified from progenitors, and by determining the extent of lineage plasticity between AT1s and AT2s during alveolar development. Aim 2 will define the functional role of the transcription cofactor, Hopx, in AT1 lineage specification during alveolar development by determining the phenotypic effects of loss of Hopx on the AT1 lineage and by elucidating changes in AT1 gene expression associated with loss of Hopx. Consistent with its role as a transcriptional co-repressor, Hopx is likely to repress AT2 fate by affecting AT2 gene transcription specifically in AT1s. Together, these approaches will determine the temporal control of AT1 lineage specification and elucidate a mechanism by which Hopx may drive AT1 lineage specification through repression of an AT2 transcriptional program, and, consequently, repression of AT2 cell fate.